Gene expression in prokaryotes and eukaryotes is a highly regulated process. Inappropriate expression (over-expression or under-expression) of “normal” or “healthy” genes is associated with many diseases and disease processes. Similarly, expression of mutated genes is also associated with many diseases. Controlling the expression of these genes is one of the ways through which diseases can be treated.
All genes contain transcriptional regulatory sequences upstream and downstream from the transcription start site. Transcription factors recognize and bind to transcriptional regulatory sequences and control the production of message transcribed from the gene. Transcriptional regulatory nucleic acid sequences involved in the regulation of gene expression include promoters, enhancers, and regulatory sequences to which transcription factors or transcriptional regulatory proteins bind, which are required for initiation of transcription. Although transcriptional regulatory sequences are most frequently found just upstream of the transcription start site, they can also be found much further upstream, or on the 3′ of the gene, or within the introns and exons that make up a gene.
A promoter is a region in a DNA sequence generally 1 to about 100 or 200 basepairs upstream of the transcription start site of a gene and typically contains or is adjacent to one or more transcription factor binding sites. An enhancer is a region in a DNA sequence that generally functions to increase transcription of a gene under its control. Enhancers are found upstream and/or downstream from the transcription start site. Enhancers can be located hundreds or even thousands of basepairs away from the transcription start site. Transcription factors bind to promoters and enhancers to regulate transcription.
The sequences of numerous transcriptional regulatory sequences are known in the art, some of which can be found in the “Eukaryotic Promoter Database” developed and maintained by members of the Bioinformatics Group of the ISREC (Swiss Institute for Experimental Cancer Research), which is avalable on the Internet. However, absent a thorough analysis of the function of particular sequences found within a given promoter or enhancers, it is impossible to determine whether the particular sequences are important in regulating gene transcription. Once transcriptional regulatory sequences have been identified, they may be utilized to regulate expression of the endogenous genes and may be incorporated into heterologous nucleic acid constructs for use in regulated expression of transgenes. Accordingly, it is of interest to identify and characterize the transcriptional regulatory regions of genes. Of particular interest are the regulatory regions of genes associated with various disease conditions, examples of which are described below.
Mammalian cyclin D1 (CCND1, also named PRAD1 or BCL1) has applications to a number of cancers including but not limited to breast cancers, colon cancers and pancreatic cancers, and plays a critical role in regulating the GOES checkpoint of the cell cycle of normal mature animal cells. (See Sherr, 1996)
CD40L ligand (CD40L) (also referred to as gp39, CD154, TRAP or T-BAM) plays a critical role in T cell dependent humoral immune responses by interacting with CD40, which provides a signal needed for T cell activation and recognition of antigen-MHC complexes by the T cell receptor.
Viral induced Hepatitis B (HBV) in humans is estimated to have infected 300 million people worldwide, with a small but significant number of infected individuals developing severe pathologic consequences, including chronic hepatic insufficiency, cirrhosis, and hepatocellular carcinoma. HBV-specific promoters involved in viral replication are therefore relevant to both therapy of HBV disease and regulated gene expression which is specific to liver cells.
The vancomycin resistance enzyme VanH has been associated with the recently observed increase in the incidence of infection and colonization with vancomycin-resistant enterococci (VRE). Therefore, regulated expression of VanH is relevant to treatment of VRE.
Prostate cancer is the most frequently diagnosed cancer in males in the United States. Current treatments for metastatic prostate cancer involve targeting the androgen receptor (AR) using surgical or chemical means. Regulated expression of the androgen receptor is relevant to treatment of prostate cancer.
Her2 (human epidermal growth factor receptor2; c-erbB2, neu) is a tyrosine kinase growth factor receptor which is overexpressed by breast cancer cells, ovarian cancer cells and a variety of other cancer cells. Accordingly, regulated expression of Her-2 is relevant to modulating such overexpression.
The β-lactamase gene confers ampicillin resistance to E. coli. Accordingly, regulated expression of β-lactamase is relevant to modification of such antibiotic resistance.
The present invention provides the sequences of the transcriptional regulatory regions of genes associated with various disease conditions together with a functional characterization of such sequences.